The present invention provides human tyrosine-DNA phosphodiesterases (TDPs). In particular, the present invention provides novel recombinant nucleic acids and proteins, including mutant TDPs, vectors, and TDP-producing cells, as well as co-factors for enzyme activity. The present invention further provides methods for high through-put enzymatic assay systems utilizing the TDPs of the present invention.
Cellular DNA is subjected to constant attack by reactive free radicals, metabolites that can act as alkylating agents produced within cells and DNA damaging agents present in environment (e.g., UV light and cytotoxic compounds like camptothecin). In humans, cells have developed various sophisticated mechanisms involving at least 130 DNA repair gene products to sense and correct DNA damage, in order to minimize toxic and mutagenic consequences and to preserve the integrity of genome (Pouliot et al., Science 286:552 [1999]). Various enzymes work in concert to achieve this goal.
Topoisomerases are cellular enzymes that are crucial for replication and transcription of the genome. Topoisomerases work by cleaving the DNA backbone, thereby allowing the topological changes needed for DNA replication and transcription to occur. After these processes have been completed, topoisomerases reseal the DNA backbone (Wang, Ann. Rev. Biochem., 65:635 [1996]). In some aspects, topoisomerases work efficiently, as DNA breakage is accompanied by covalent binding between the enzyme and the DNA, to create an intermediate structure that is resolved during the resealing step. However, this mechanism also makes topoisomerases potentially dangerous. For example, if the resealing step fails, a normally transient break in DNA becomes a long-term disruption, one with a topoisomerase covalently joined to it. Unless a means to restore the continuity of the DNA is provided, the affected cell will die.
In virtually all topoisomerases, the heart of the covalent complex is a phosphodiester bond between a specific tyrosine residue of the enzyme and one end of the break (i.e., the 3xe2x80x2 end for eukaryotic topoisomerase I and the 5xe2x80x2 end for topoisomerases II and III). The high-energy nature of this bond normally ensures that the resealing step occurs.
Failure of the resealing step is dramatically increased by various drugs, including camptothecin. Camptothecin has been considered to be a promising anti-cancer agent because it specifically targets eukaryotic topoisomerase I (Chen et al., Ann. Rev. Pharmacol. Toxicol., 94:194 [1994]). Protein-linked breaks also accumulate when topoisomerases act on DNA containing structural lesions like thymine dimers, abasic sites and mismatched base pairs (Pommier et al., Biochem. Biophys. Acta 1400:83 [1998]). To the extent that such lesions arise during the normal cell lifespan, topoisomerase-associated damage may be unavoidable.
Repair of topoisomerase-DNA covalent complexes is important to the cell. However, the means involved in this repair are not well understood. Hydrolysis of the bond joining the topoisomerase to DNA has been proposed as a way to effect release of the topoisomerase such that the cleaved DNA could undergo conventional modes of break repair (See, e.g., Friedberg et al., DNA Repair and Mutagenesis, ASM Press, Washington, D.C. [1995]; Kanaar et al., Trends Cell. Biol., 8:483 [1998]). Although no such hydrolysis has been reported for covalent complexes between DNA and topoisomerases II or III, hydrolysis has been described for covalent complexes between DNA and topoisomerase I (Yang et al., Proc. Natl. Acad. Sci. USA 93:11534-11539 [1996]). Thus, there remains a need in the art to provide means to repair cellular damage, including that caused by abnormal DNA replication. Indeed, an understanding of topoisomerases and their functions is needed in order to develop means for use of topoisomerases as targets for cancer therapy or anti-aging processes.
The present invention provides human tyrosine-DNA phosphodiesterases (TDPs). In particular, the present invention provides novel recombinant nucleic acids and proteins, including mutant TDPs, vectors, and TDP-producing cells, as well as co-factors for enzyme activity. The present invention further provides methods for high through-put enzymatic assay systems utilizing the TDPs of the present invention.
In some embodiments, the present invention provides single and double-stranded nucleic acid sequences encoding human TDP. In other embodiments, the present invention provides polypeptides comprising wild-type, as well as mutant TDPs. In some preferred embodiments, the mutant polypeptides have deletions of the amino-terminal 39 or 174 amino acids and end at residues 522 or 545.
In other embodiments, the present invention provides vectors comprising nucleic acid sequences encoding wild-type and mutant TDPs. In particularly preferred embodiments, the TDPs are human. In some preferred embodiments, the vectors of the present invention are contained within host cells. In some particularly preferred embodiments, the vectors of the present invention contained within host cells express wild-type and/or mutant TDPs (i.e., the vectors are xe2x80x9cexpression vectorsxe2x80x9d). In alternative preferred embodiments, the expressed TDPs are human.
In further embodiments, the present invention provides methods for protein purification and refolding that result in the production of soluble wild-type or mutant human TDP polypeptides. In some embodiments, the soluble human TDP polypeptides are used in crystal structure determinations, while in other embodiments, the TDP polypeptides find use in high through-put screening methods.
In some embodiments, the high through-put screening methods of the present invention provide means to identify and characterize compounds capable of inhibiting, stimulating, or otherwise modulating TDP and/or its function. In some preferred embodiments, p-nitrophenyl thymidine-3xe2x80x2-phosphate free acid is used as a chromogenic substrate and Mn++ (manganese cation) is used as cofactor to provide enzymatic assay systems to assess the activity of TDP and the efficacy of TDP-inhibitors.